To understand mammalian developmental mechanisms at the molecular level, it is necessary to identify and isolate developmental genes. In the mouse, mutations that result in developmental abnormalities have identified a number of candidate developmental genes. However, cloning genes identified by spontaneous or chemically induced mutations is technically difficult and has been accomplished only rarely. A powerful and direct method to induce a mutation and molecularly clone the mutated gene is insertional mutagenesis. Insertional mutations result from the integration of foreign DNA into a gene. The integrated sequence serves a dual purpose. It not only creates the mutation but also acts as a molecular marker "tagging" the locus. By recloning the integrated sequence the affected gene can be easily recovered as well. Insertional mutagenesis occurs spontaneously in the mouse due to endogenous retroviral infection of embryos or germ cells; and through experimental introduction of DNA or retrovirus to produce transgenic mice. Approximately 10% of proviral integrations or transgenic DNA insertions are into genes, resulting in mutations. We have analyzed 2 transgenic strains derived from embryonic stem (ES) cells infected with a retroviral vector for insertional mutations. These strains are unique in that they each carry multiple, independently integrated proviral sequences. The 413 strain segregates 4 proviruses and the 412 strain, 23. Systematic genetic and molecular analysis has allowed us to determine that 5 of the 27 proviruses are associated with mutations. We are now carrying out molecular and phenotypic studies on these mutants.